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Three-Dimensional Spheroid Culture Increases Exosome Secretion from Mesenchymal Stem Cells

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Tissue Engineering and Regenerative Medicine Aims and scope

Abstract

Background:

Mass production of exosomes is a prerequisite for their commercial utilization. This study investigated whether three-dimensional (3D) spheroid culture of mesenchymal stem cells (MSCs) could improve the production efficiency of exosomes and if so, what was the mechanism involved.

Methods:

We adopted two models of 3D spheroid culture using the hanging-drop (3D-HD) and poly(2-hydroxyethyl methacrylate) (poly-HEMA) coating methods (3D-PH). The efficiency of exosome production from MSCs in the 3D spheroids was compared with that of monolayer culture in various conditions. We then investigated the mechanism of the 3D spheroid culture-induced increase in exosome production.

Results:

The 3D-HD formed a single larger spheroid, while the 3D-PH formed multiple smaller ones. However, MSCs cultured on both types of spheroids produced significantly more exosomes than those cultured in conventional monolayer culture (2D). We then investigated the cause of the increased exosome production in terms of hypoxia within the 3D spheroids, high cell density, and non-adherent cell morphology. With increasing spheroid size, the efficiency of exosome production was the largest with the least amount of cells in both 3D-HD and 3D-PH. An increase in cell density in 2D culture (2D-H) was less efficient in exosome production than the conventional, lower cell density, 2D culture. Finally, when cells were plated at normal density on the poly-HEMA coated spheroids (3D-N-PH); they formed small aggregates of less than 10 cells and still produced more exosomes than those in the 2D culture when plated at the same density. We also found that the expression of F-actin was markedly reduced in the 3D-N-PH culture.

Conclusion:

These results suggested that 3D spheroid culture produces more exosomes than 2D culture and the non-adherent round cell morphology itself might be a causative factor. The result of the present study could provide useful information to develop an optimal process for the mass production of exosomes.

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Acknowledgement

This research was supported by a grant from the Korea Health Technology R&D Project funded by the Ministry of Health & Welfare, Republic of Korea (HI17C2191).

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Authors and Affiliations

  1. Department of Molecular Sciences and Technology, Ajou University, 206, World cup-ro, Yeongtong-gu, Woncheon-dong, Suwon, 16499, Republic of Korea

    Mijin Kim, Hee-Woong Yun & Byoung-Hyun Min

  2. Cell Therapy Center, Ajou University Medical Center, 206 World Cup-ro, Yeongtong-gu, Suwon, 16499, Republic of Korea

    Mijin Kim, Hee-Woong Yun & Byoung-Hyun Min

  3. Department of Orthopedic Surgery, School of Medicine, Ajou University, 206 World Cup-ro, Yeongtong-gu, Suwon, 16499, Republic of Korea

    Do Young Park & Byoung-Hyun Min

  4. Department of Biomedical Sciences, Inha University College of Medicine, 100 Inha-ro, Nam-gu, Incheon, 22212, Republic of Korea

    Byung Hyune Choi

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  1. Mijin Kim
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  2. Hee-Woong Yun
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Correspondence to Byung Hyune Choi or Byoung-Hyun Min.

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Figure S1

Comparison of the amount of exosomes secreted from hBM-MSCs primed with various stimuli. hBM-MSCs were cultured in monolayer at normal density (2D) and were untreated (Control) or treated respectively with IL-1β, TNF-α, Poly I:C, and 3D-HD. The amounts of exosomes obtained from 1 × 105 cells are presented for each group at 3 days of culture. Data are presented as the mean and SD from three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 by one-way ANOVA. 3D, three-dimensional; hBM-MSCs; human bone marrow-mesenchymal stem cells; 2D, two-dimensional; 3D-HD, 3D spheroids formed by the hanging drop method; ANOVA, analysis of variance; IL-1β, interleukin 1 beta; TNF-α, tumor necrosis factor alpha; Poly I:C, polyinosinic-polycytidylic acid. (JPEG 123 kb)

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Kim, M., Yun, HW., Park, D.Y. et al. Three-Dimensional Spheroid Culture Increases Exosome Secretion from Mesenchymal Stem Cells. Tissue Eng Regen Med 15, 427–436 (2018). https://doi.org/10.1007/s13770-018-0139-5

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  • DOI: https://doi.org/10.1007/s13770-018-0139-5

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